In FY 2019, we contributed to four (4) original research manuscripts on this topic as follows: Manuscript #1: Title: Cytokine Diversity in Human Peripheral Blood Eosinophils: Profound Variability of IL-16. Eosinophilic leukocytes maintain homeostasis and promote dysfunction via release of preformed immunomodulatory mediators. Here we explored human eosinophil heterogeneity with a specific focus on naturally occurring variations in cytokine content. We found that human eosinophil-associated cytokines varied on a continuum from minimally (CV < 50%) to moderately variable (50% < CV 90%). Within the moderately variable group, we detected immuno-reactive IL-27, a mediator not previously associated with human eosinophils. However, our major finding was the profound variability of eosinophil-associated IL-16 (CV = 103%). Interestingly, eosinophil IL-16 content correlated directly with body mass index (R-squared = 0.60, ***p < 0.0001) in one donor subset. We found no direct correlation between eosinophil IL-16 content and donor age, sex, total leukocytes, lymphocytes, or eosinophils (cells per microliter), nor was there any relationship between IL-16 content and the characterized -295T/C IL-16 promoter polymorphism. Likewise, although eosinophil IL-1alpha, IL-1beta, and IL-6 levels correlated with one another, there was no direct association between any of these cytokines and eosinophil IL-16 content. Finally, a moderate increase in total dietary fat resulted in a 2.7-fold reduction in eosinophil IL-16 content among C57BL/6-IL5tg mice. Overall, these results suggest that relationships between energy metabolism, eosinophils, and IL-16 content are not direct or straightforward. Nonetheless, given our current understanding of the connections between asthma and obesity, these findings suggest important eosinophil-focused directions for further exploration. Critical point: Eosinophil-associated cytokines vary on a continuum from minimally to moderately variable. By contrast, eosinophil IL-16 content varied profoundly and via a unique pattern with no correlations to levels of other eosinophil-derived cytokines. Given our current understanding of the links between eosinophils, asthma, and obesity, an improved understanding of eosinophils and IL-16 content may provide important connections worthy of further exploration. Ref: Ma M, Percopo CM, Sturdevant DE, Sek AC, Komarow HD, Rosenberg HF. 2019. J Immunol. 203:520-531. Manuscript #2: Title: Eosinophils Do Not Drive Acute Muscle Pathology in the mdx Mouse Model of Duchenne Muscular Dystrophy Eosinophils are present in muscle lesions associated with Duchenne muscular dystrophy and dystrophin-deficient mdx mice that phenocopy this disorder. Although it has been hypothesized that eosinophils promote characteristic inflammatory muscle damage, this has not been fully examined. In this study, we generated mice with the dystrophin mutation introduced into PHIL, a strain with a transgene that directs lineage-specific eosinophil ablation. We also explored the impact of eosinophil overabundance on dystrophinopathy by introducing the dystrophin mutation into IL-5 transgenic mice. We evaluated the degree of eosinophil infiltration in association with myofiber size distribution, centralized nuclei, serum creatine kinase, and quantitative histopathology scores. Among our findings, eosinophils were prominent in the quadriceps muscles of 4-wk-old male mdx mice but no profound differences were observed in the quantitative measures of muscle damage when comparing mdx versus mdx.PHIL versus mdx.IL5tg mice, despite dramatic differences in eosinophil infiltration (CD45+CD11c-Gr1-MHC class II-loSiglecF+ eosinophils at 1.2 0.34% versus <0.1% versus 20 7.6% of total cells, respectively). Further evaluation revealed elevated levels of eosinophil chemoattractants eotaxin-1 and RANTES in the muscle tissue of all three dystrophin-deficient strains; eotaxin-1 concentration in muscle correlated inversely with age. Cytokines IL-4 and IL-1R antagonist were also detected in association with eosinophils in muscle. Critical point: Our findings challenge the long-held perception of eosinophils as cytotoxic in dystrophin-deficient muscle; we show clearly that eosinophil infiltration is not a driving force behind acute muscle damage in the mdx mouse strain. Ongoing studies will focus on the functional properties of eosinophils in this unique microenvironment. Ref: Sek AC, Moore IN, Smelkinson MG, Pak K, Minai M, Smith R, Ma M, Percopo CM, Rosenberg HF. 2019. J Immunol. 203:476-484 Manuscript #3: Title: Impact of eosinophil-peroxidase (EPX) deficiency on eosinophil structure and function in mouse airways. Eosinophil peroxidase (EPX) is a major constituent of the large cytoplasmic granules of both human and mouse eosinophilic leukocytes. Human EPX deficiency is a rare, autosomal-recessive disorder limited to the eosinophil lineage. Our intent was to explore the impact of EPX gene deletion on eosinophil content, structure, and function. In response to repetitive intranasal challenge with a filtrate of the allergen, Alternaria alternata, we found significantly fewer eosinophils peripherally and in the respiratory tracts of EPX-/- mice compared to wild-type controls; furthermore, both the major population (Gr1-/lo ) and the smaller population of Gr1hi eosinophils from EPX-/- mice displayed lower median fluorescence intensities (MFIs) for Siglec F. Quantitative evaluation of transmission electron micrographs of lung eosinophils confirmed the relative reduction in granule outer matrix volume in cells from the EPX-/- mice, a finding analogous to that observed in human EPX deficiency. Despite the reduced size of the granule matrix, the cytokine content of eosinophils isolated from allergen-challenged EPX-/ - and wild-type mice were largely comparable to one another, although the EPX-/- eosinophils contained reduced concentrations of IL-3. Other distinguishing features of lung eosinophils from allergen-challenged EPX-/- mice included a reduced fraction of surface TLR4+ cells and reduced MFI for NOD1. Interestingly, the EPX gene deletion had no impact on eosinophil-mediated clearance of gram-negative Haemophilus influenzae from the airways. Critical point: Although no clinical findings have been associated with human EPX deficiency, our findings suggest that further evaluation for alterations in eosinophil structure and function may be warranted. Ref: Percopo CM, Krumholz JO, Fischer ER, Kraemer LS, Ma M, Laky K, Rosenberg HF. 2019. J Leukoc Biol. 105:151-161 Manuscript #4: Title: Aspergillus fumigatus secreted alkaline protease 1 (Alp1) mediates airways hyper-responsiveness in severe asthma. The hallmark features of allergic asthma are type 2 (eosinophilic) inflammation and airways hyper-responsiveness (AHR). While these features often co-manifest in mouse lungs in vivo, we demonstrate here that the serine protease Alp1 from the ubiquitous mold and allergen, Aspergillus fumigatus (Af), can induce AHR in mice unable to generate eosinophilic inflammation. Strikingly, Alp1 induced AHR in mice devoid of protease activated receptor 2/F2 trypsin like receptor 1 (PAR2/F2RL1), a receptor expressed in lung epithelium that is critical for allergic responses to protease-containing allergens. Instead, using precision-cut lung slices (PCLS) and human airway smooth muscle (ASM) cells, we demonstrate that Alp1 directly increased contractile force. Critical point: Taken together, these findings suggest that Alp1 induces bronchoconstriction through mechanisms that are largely independent of allergic inflammation and point to a new target for direct intervention of fungal-associated asthma. Ref: Redes JL, Basu T, Ram-Mohan S, Ghosh CC, Chan EC, Sek AC, Zhao M, Krishnan R, Rosenberg HF. 2019. Immunoho